Characterization of plasmids carrying blaCTX-M genes among extra-intestinal Escherichia coli clinical isolates in Ethiopia

CTX-Ms are encoded by blaCTX-M genes and are widely distributed extended-spectrum β-lactamases (ESBLs). They are the most important antimicrobial resistance (AMR) mechanism to β-lactam antibiotics in the Enterobacteriaceae. However, the role of transmissible AMR plasmids in the dissemination of blaCTX-M genes has scarcely been studied in Africa where the burden of AMR is high and rapidly spreading. In this study, AMR plasmid transmissibility, replicon types and addiction systems were analysed in CTX-M-producing Escherichia coli clinical isolates in Ethiopia with a goal to provide molecular insight into mechanisms underlying such high prevalence and rapid dissemination. Of 100 CTX-Ms-producing isolates obtained from urine (84), pus (10) and blood (6) from four geographically distinct healthcare settings, 75% carried transmissible plasmids encoding for CTX-Ms, with CTX-M-15 being predominant (n = 51). Single IncF plasmids with the combination of F-FIA-FIB (n = 17) carried the bulk of blaCTX-M-15 genes. In addition, IncF plasmids were associated with multiple addiction systems, ISEcp1 and various resistance phenotypes for non-cephalosporin antibiotics. Moreover, IncF plasmid carriage is associated with the international pandemic E. coli ST131 lineage. Furthermore, several CTX-M encoding plasmids were associated with serum survival of the strains, but less so with biofilm formation. Hence, both horizontal gene transfer and clonal expansion may contribute to the rapid and widespread distribution of blaCTX-M genes among E. coli populations in Ethiopian clinical settings. This information is relevant for local epidemiology and surveillance, but also for global understanding of the successful dissemination of AMR gene carrying plasmids.


Acquisition of MDR.
We had previously assessed multiple drug resistance profiles among the parental clinical isolates used in this study 11 (Supplementary Table S1). To address whether this characteristic could be conferred to recipient bacteria through the transmissibility of AMR plasmids, we performed an antimicrobial susceptibility test using the disk diffusion method on all 75 parental clinical isolates and their corresponding recipients of AMR plasmids. The 75 isolates were 100% resistant to cefotaxime, ceftazidime and cefepime confirming our earlier finding 11 . Among second-generation plasmid recipient strains this resistance rate remained 100% for cefotaxime and dropped slightly to 90.7% and 88.0% for ceftazidime and cefepime respectively (Fig. 1). The resistance rates for ciprofloxacin, sulfamethoxazole/trimethoprim amoxicillin-clavulanic acid, gentamicin, cefoxitin, amikacin and meropenem were 92.0%, 88.0%, 72.0%, 50.7%, 17.3%, 1.3% and 1.3%, respectively (Fig. 1). Among the AMR plasmid recipient strains, resistance rates to these non-β-lactams antibiotics were 48%, 65.3%, 41.3% and 1.3% for ciprofloxacin, sulfamethoxazole/trimethoprim, gentamicin, and amikacin respectively (Fig. 1). Moreover, 56% of the second-generation strains were resistant to the β-lactamase inhibitor amoxicillin-clavulanic acid and 9.3% were resistant to cefoxitin, whereas no strain acquired resistance to meropenem (Fig. 1).
AMR plasmid replicon types. To our knowledge, the biology of AMR plasmids harboured by clinical bacterial isolates from Ethiopia has not been studied. We began this important work by focusing first on identifying Table 1. The transmissibility of 100 CTX-M producing clinical E. coli isolates included in this study. a Determination of the distribution of the isolates among the six phylogenetic groups was performed in our previous study 11 . Numbers in parentheses indicate the number of isolates belonging to a phylogroup that were selected for use in this study. The geographical origin of these strains can be identified from information provided in supplementary Table S1. b Numbers in parentheses that are italicised indicate B2 isolates of international high-risk clone ST131. c The degree of transmissibility of bla CTX-M-15 , other group 1 bla CTX-Ms , and group 9 bla CTX-Ms is 68% (51 of 75), 22.7% (17/75) and 9.3% (7/75), respectively.  Table 2. The distribution of alternative β-lactamases among CTX-M producing clinical E. coli isolates and recipient strains. a Determination of the distribution of the isolates among the six phylogenetic groups is presented as in Table 1 and derived from our previous study 11 . b Observe that the total number of parental strains positive for TEM (n = 13) and TEM-OXA (n = 39) is 52. www.nature.com/scientificreports/ frequency of 7.8%, and the I1-Iγ replicon type at 6.3% (Fig. 2). The other types identified were detected at below 5% frequency in the plasmid recipient strains (Fig. 2). We also determined the association of replicon types with antibiotic resistance genes. Out of the 51 transmissible plasmids having bla CTX-M-15 (see Table 1), 45 (88.2%) were typed using PBRT. Among these 45 PBRT-typed transmissible plasmids, 35 (77.8%) carried combinations of Inc replicon types while the remaining 10 (22.2%) carried just a single replicon type (Table 3). The combination of F-FIA-FIB plasmid replicon was frequently associated with other group-1 CTX-M types encoded by bla CTX-M-101 , bla CTX-M-103 , bla CTX-M-142 , bla CTX-M-180 , bla CTX-M-182 and bla CTX-M-225 (Table 3). In addition, 6 plasmids carrying group-9 bla CTX-M-27 belonged to 3 different replicon types consisting of a single IncF (n = 1), as well as in apparent combination with F-FIA-FIB (n = 4), or with F-FIA-FIB, and I1-Iγ (n = 1) ( Table 3).
Addiction systems for plasmid maintenance. Plasmid maintenance during host replication is a vital aspect of transmissibility. We investigated the presence of eight plasmid encoded addiction systems according to a previously described PCR-based detection system 19 . Six plasmid addiction system types (pemKI, ccdAB, vagCD, hok-sok, pndAC and srnBC) could be identified among the 75 parental isolates (Table 4) and the corresponding plasmid recipient strains (Table 5). In the parental strains, the 337 plasmid addition system combinations detected were pemKI (n = 72), srnBC (n = 68), ccdAB (n = 68), vagCD (n = 55), pndAC (n = 48), and hok-sok Table 3. Distribution and number of plasmid replicon types and associated addiction systems in recipient E. coli containing transmissible plasmids categorised according to CTX-M carriage and plasmid replicon type. a Refers to the total number of isolates that had an identifiable addiction system encoded on a plasmid with defined replicon type. b Refers to the average number of addiction systems encoded on a plasmid with defined replicon type; calculated as 'total' number of identified addiction systems divided by 'n' number of isolates containing a plasmid(s) with stated replicon type.  www.nature.com/scientificreports/ Table 4. Distribution of addiction systems among transmissible plasmids encoding CTX-M genes and harboured by donor parental strains of clinical origin in Ethiopia. a Refers to the total number of isolates that had an identifiable addiction system encoded on a plasmid with defined CTX-M producing genes alone or in combination with alternative β-lactamase-encoding genes (non-ESBLs genes). b Refers to the average number of addiction systems encoded on a plasmid with defined β-lactamase-encoding genes; calculated as 'total' number of identified addiction systems divided by 'n' number of isolates containing a plasmid(s) with stated β-lactamase-encoding gene.   Table 5. Distribution of addiction systems among CTX-M-encoding transmissible plasmids that were successfully mobilised to recipient E. coli a . a Mobilisation of transmissible plasmids into E. coli J53 AziR was by conjugation and into HB101 was by chemical transformation. b Refers to the total number of isolates that had an identifiable addiction system encoded on a plasmid with defined CTX-M producing genes alone or in combination with alternative β-lactamase-encoding genes (non-ESBLs genes). c Refers to the average number of addiction systems encoded on a plasmid with defined β-lactamase-encoding genes; calculated as 'total' number of identified addiction systems divided by 'n' number of isolates containing a plasmid(s) with stated β-lactamase-encoding gene. www.nature.com/scientificreports/ (n = 26). The relBE and parDE plasmid addiction systems were not detected in the parental E. coli strains analysed. On the other hand, in the plasmid recipient strains, a total of 176 plasmid addiction system combinations were identified consisting of pemKI (n = 47), srnBC (n = 42), ccdAB (n = 39), vagCD (n = 21), pndAC (n = 23), and hok-sok (n = 4). Once again, none of the strains harboured the relBE and parDE plasmid addiction systems. There was direct correlation between the combination of plasmid replicon types and the mean numbers of addiction systems detected ( Table 3). The highest mean numbers of addiction systems were observed in plasmids with a combination of four replicon types. In contrast, the lowest mean numbers of addiction systems correlated to plasmids with a single Inc replicon type. However, no clear correlation between any of the mean numbers of plasmid addiction system combinations and any β-lactamase type including the CTX-M in either the donor parental strains (Table 4) or the recipient strains (Table 5) could be identified.

CTX-M with other β-lactamases n
Plasmid transmissibility and biofilm formation. Knowledge of the interplay between bacterial biofilms and plasmids could benefit the development of therapeutic measures to control antimicrobial resistance plasmid transmissibility. Hence, we compared the ability of 12 donor parent strains and the corresponding plasmid recipient counterparts for their ability to form biofilms in a microtiter plate assay. The criteria for selecting this subset were: (1) a primary focus on the B2 phylotype because these are usually extra-intestinal bacteria, (2) a primary focus on the ST131 international high risk clone, (3) a spread of isolates having one to multiple plasmid replicon types, (4) a spread of isolates having one to multiple CTX-M types, and (5) Table S1). Only two donor parental 'P' strains could be classified as a strong biofilm former-P106, or moderate biofilm former-P107 (Fig. 3). The remaining donor parental strains were either weak biofilm formers (P2, P3, P22, P154, P163, P174, and P184), or failed to form biofilms (P9, P74 and P149) under the experimental conditions tested (Fig. 3). Interestingly, only the plasmids transmissible from P22, P154 and P184 plasmid could confer to the recipient 'R' strains (R22, R154 and R184) the ability to form any degree of biofilm (Fig. 3). The replicon types identified in these three isolates were F-FIB, F-FIA-FIB and F-FIA-FIB, respectively (Supplementary Table S1). Hence, we could identify just three cases where the P22, P154 and P184 derived AMR plasmid(s) may have captured plasmid-encoded biofilm promoting factors.
Influence of plasmid carriage on serum resistance. Since carriage of AMR plasmids influence the extent of serum resistance 16 , we compared the ability of a selected group of donor parent strains and the corresponding plasmid recipient counterparts for their ability to confer resistance to normal human serum. Ten of the isolates selected above were also used in this serum sensitivity study (Supplementary Table S1). Two parental 'P' isolates (P9 and P74) and four recipient 'R' strains (R2, R9, R74 and R106) were totally sensitive to prolonged exposure to human serum (Fig. 4). This was comparable to the serum sensitive phenotype of the control strain  Table S1). In contrast, serum resistance in the strains P2, and P106 is not transmissible and must be associated with chromosomal encoded elements or elements encoded on nontransmissible plasmids. Figure 3. Biofilm formation efficiency of pathogenic E. coli strains, isolated from Ethiopian patients. Data was generated from a minimum of three biological and three technical replicates for every isolate and plotted using GraphPad-5.0. One-way ANOVA with inbuilt Tukey's multiple comparison test was applied to calculate statistically significance between control strain E. coli J53 (black bar) and Parental strains ('P' , dark grey bars) and respective recipient strains ('R' , light grey bar). P < 0.0001: ***P < 0.001: **P < 0.01: *P > 0.05: non-significant (ns).

Discussion
This study is the first to report on plasmid transmissibility, replicon types and associated addiction systems among CTX-M-producing MDR E. coli clinical isolates from Ethiopia (Supplementary Table S1). It is also the first study from Ethiopia that describes these plasmid replicon types in association with the clonal distribution of CTX-M-producing E. coli clinical isolates. The data verify the long-held notion that horizontal gene transfer is a major contributor to the clonal expansion and widespread distribution of CTX-M-encoding genes in Ethiopia. These findings are alarming for it demonstrates that plasmid transmission is a major factor in the co-transfer of genes encoding resistance to non-cephalosporins antimicrobials among bacterial populations in Ethiopia. Consequently, in the absence of any intervention rapid spread of multiple drug resistance among bacterial populations in clinical, agricultural and community settings will continue unabated. An uneasy solace is for last resort carbapenem drugs, such as meropenem, where the isolates were still highly susceptible. Possible resistance to colistin, which is a last-resort treatment for MDR Gram-negative infections, was not tested because it was not approved for clinical use in Ethiopia at the time of the study. While 75% of the isolates examined contained CTX-M genes on transmissible plasmids, the remaining 25% of isolates were unable to transfer these genes to the E. coli recipient. Moreover, the alternative β-lactamase bla SHV was also non-transmissible. These non-transferable bla CTX-M and bla SHV genes are likely to be integrated into the host chromosome or be present on non-transmissible plasmids. These isolates are worthy of further genetic characterization as it may provide some new clues on the increased genetic heterogeneity among the markers for MDR and their dissemination. A precedent for this type of novel discovery is the chromosomal location of bla CTX-M-15 in the very extensive AMR and virulent subclone of ST131 H30Rx 20 . This origin was due to the mobilization of a plasmid-located Tn3-like ISEcp1-bla CTX-M-15-orf477 element that subsequently integrated into the bacterial genome. ISEcp1 is an IS that contributes to the effective capture, expression, and mobilization of AMR genes from multiple sources, and is commonly located in the upstream of region of the CTX-M encoding genes 21,22 . Our observation of ISEcp1 in 97.3% of parental strains and 94.7% of the corresponding recipient strains corroborates these earlier findings.
Although exact genetic associations could not be directly determined without plasmid sequencing data, 59 (78.7%) transferrable bla CTX-M genes appeared to be located on narrow-host-range IncF plasmids. This agrees with IncF plasmids being the major carrier of bla CTX-M genes 23 . IncF plasmids encoding for CTX-Ms are detected in a range of E. coli sequence types 24 , but have a particularly strong association with the global spread of CTX-M-15 producing E. coli ST131 23 . IncF plasmids contribute various AMR determinants and virulence associated factors that create competitive fitness advantages that select for the success of the ST131 clone 25 , and the evolution of ST131 sub-lineages such as H30, which include H30R1 and H30Rx 7,25,26 . Consistent with this profile is our observation of IncF plasmids harboured by isolates associated with a variety of E. coli sequence type lineages, including the international high risk E. coli ST131 lineage. This indicates that the widespread prevalence of CTX-M-15 encoding genes in Ethiopia is facilitated by both host cell clonal expansions and horizontal gene transfer by IncF plasmids. We also identified IncF plasmids in E. coli phylogenetic groups primarily considered to be commensal E. coli, corroborating an earlier claim 27 .
We suspect that the IncF plasmids identified in this study have either single replicons or multiple replicons. The different combinations can reflect the fusion between different types creating a replicon chimera, or that multiple plasmids simultaneously coexist in the same cell 18,24,28,29 . This phenomenon is quite helpful to establish and trace relevant epidemiologic plasmid lineages. However, the fitness advantages conferred by a plasmid  ) and their corresponding recipient strains (light grey bars) and the control strain J53 (black bar) after exposure to active human serum for 0 and 3 h. The susceptibility to killing was calculated as follows: log kill = (log10 CFU per milliliter of initially added bacteria-0 h)-(log10 CFU per milliliter of bacteria surviving the incubation after 3 h). GraphPad-5.0 was used to plot data from a minimum of two biological replicates of every isolate. Means and standard errors of the results are shown. One-way ANOVA with inbuilt Tukey's multiple comparison test was applied to calculate statistical significance between the corresponding parental and recipient strains. P < 0.0001: ***P < 0.001: **P < 0.01: *P > 0.05: non-significant (ns). www.nature.com/scientificreports/ having multiple replicons within the same incompatibility group is not clear, since it surely would raise issues of replication coordination, regulation, and instability brought about by incompatibility phenomena. This contrasts with plasmids co-opting replicons from different incompatibility groups, which would extend replication opportunities within diverse hosts. Hence, follow-up work focused on whole genome or plasmid sequencing should assess if the IncF plasmid replicons represent discrete and intact genetic entities, or whether they are merely fusions between different types creating replicon chimeras, as has been previously reported 18 . Assessing replicon functionality is also warranted. Interestingly, from eight addiction system types analysed in this study, we could detect three type I and three type II addiction systems. In the parental donor isolates this amounted to 337 combinations of addition systems. However, just 176 addiction system combinations were detected in the recipient transconjugants. As suggested by others, the implication of these findings is that addiction systems might be positioned on a non-conjugative plasmid or in the chromosome, and those located on conjugative plasmids are associated with the transmissible bla CTX-M genes 19,30,31 . Moreover, almost all detected addiction systems in the recipient strains were carried on IncF plasmids, corroborating previous findings 32 . Plasmid addiction systems play important roles in plasmid stability and maintenance in a bacterial population 33 , and can enhance bacteria fitness under adverse environmental conditions 34 . Hence, our data indicate that IncF plasmids use multiple addiction systems for maintenance and stability during horizontal dissemination of bla CTX-M genes within Ethiopian isolates.
An additional key element to this is the finding that these bla CTX-M harbouring plasmids also possess the possibility to disseminate other resistant genes of clinical significance, including those that confer resistance to sulfamethoxazole/trimethoprim, ciprofloxacin, amoxicillin-clavulanic acid, gentamicin, amikacin, and cefoxitin. This highlights the potential of plasmids harbouring bla CTX-M genes to quickly disseminate MDR in hospital, community, and agricultural settings. Thus, a better understanding of the origin and evolution of non-beta-lactam antibiotics resistance in Ethiopia is required.
Despite our identification and initial characterization of transmissible AMR plasmids in E. coli isolates collected at limited healthcare settings, it is evident that pivotal knowledge concerning the genetic diversity that might exist among them is still lacking. Follow up work should focus on further genetic characterization of the plasmids to better define the extent of their diversity, and to provide important information connecting plasmid backbone and replicon type with combinations of acquired multiple resistance genes and addiction modules as well as other phenotypic traits associated with pathogenicity such as serum resistance and the capability to form biofilms. Achieving this would require a method that combines S1-nuclease mediated cleavage of the plasmids, followed by pulsed field gel electrophoresis and Southern blotting 35 , and also in combination with direct plasmid sequencing, or even through whole genomic sequencing. Considering that most transmissible plasmids in this study were based on the IncF family, which in other studies has displayed extensive genetic diversity [36][37][38][39] we speculate that this would be true also for plasmids harboured in our isolate collection. Moreover, a significant percentage of transmissible plasmids possessed a non-typed replicon according to our PBRT assay. Hence, applying the more discerning genetic methods on this group of isolates will also provide new clues concerning dissemination of AMR among various E. coli populations in Ethiopia.
Apart from the IncF plasmids, we were also interested in the isolates that harboured transmissible narrowhost-range plasmids with an IncI1-Iγ or IncY replicon. Although not confirmed by sequence-based methods, the IncI1-Iγ and IncY plasmids identified in our study were often found associated with the bla CTX-M-15 gene. Interestingly, plasmids with these replicons have been detected in bacteria isolated from animals produced for food and associate with various AMR genes [40][41][42][43] . Based on this precedent, it is possible that the IncI1-Iγ and IncY plasmids identified herein may have an animal origin, which could be a source for human infections in Ethiopia. This will be confirmed in future work with Ethiopian bacterial collections expanded to include isolates from wider community and agricultural sources. The only other replicon detected in our study was IncL/M. This is a broad-host-range replicon allowing for greater transmission among diverse bacterial species, as evidenced by an association between IncL/M plasmids and bla CTX-M-3 and bla OXA-48 dissemination 44,45 .
Various E. coli pathotypes possess a variety of virulence associated factors which support their entry, colonization, survival, and dissemination within and between infected human and animal hosts. Definitive conclusions concerning pathotypes of our isolates still require sequence analysis to identify the presence of hallmark virulence genes that have been defined in earlier studies [46][47][48] . It is well established that virulence associated factors can be encoded within mobile genetic elements, such as plasmids 49 . This is also reflected in this study that revealed survivability of a subset of parental isolates and their corresponding plasmid recipient strains in normal human serum, which is attributable to specific genes carried on the CTX-M-encoding resistance plasmids. Although the data is limited, it hints to the fact that AMR plasmids from many E. coli isolates sourced in Ethiopia likely also encode for other properties that can influence lifestyle choices important for environmental survival and host pathogenicity.
We also noted that most isolates containing the bla CTX-M-14 , bla CTX-M-15 and bla CTX-M-27 genes distributed among the B2, D, and F phylo-groups. These likely represent extra-intestinal pathogenic E. coli (ExPEC) isolates, since global population studies routinely associate ExPEC bacteria within the B2 and D phylo-groups 50 . This is a serious concern given that ExPEC bacteria are a major global clinical problem 51 . Hence, we speculate a high prevalence of ExPEC in Ethiopia, although this needs to be verified on more extensive E. coli collections. This will be achievable because of our access to a diverse and expanding E. coli isolate collection via ongoing One Health laboratory-based AMR surveillance initiative in Ethiopia. The identification of virulence associated factors co-localising with plasmid-encoded AMR genes will have major ramifications for the evolution of novel and re-emerging bacterial pathogens that will pose acute health risk.
There is also interest in the isolates associated with the phylogenetic groups A, B1 and C which are unlikely to be ExPEC strains. Rather, they must be either intestinal non-pathogenic commensal isolates, or intestinal pathogenic isolates (InPEC). Either way, they have been isolated from non-stool samples, chiefly urine, suggesting www.nature.com/scientificreports/ an association with extra-intestinal infections, and which would require a translocation from the gastrointestinal tract. Since InPEC rarely caused extra-intestinal infections 52 , we suspect that the remaining extra-intestinal isolates belonging to the other phylogenetic groups A, B1 and C originated as commensal E. coli. This idea needs confirmation, but precedent comes from knowing that commensal E. coli strains can participate in extraintestinal infections when the gastrointestinal barrier is breached especially in immune-compromised patients and when the bacterial load is particularly high 53 . Our study has certain limitations. It did not directly determine which bla CTX-M genes were genetically linked with the identified plasmid replicon types. Genetic characterization of the isolates that did not transfer bla CTX-M genes was also not performed. Further, reliable genetic associations could not be directly inferred without plasmid sequencing data. Moreover, our identification and initial characterization of transmissible AMR plasmids in E. coli isolates were collected from limited healthcare settings and does not have nationwide representation. Thus, the results need confirmation in future work on collections expanded to include isolates from wider communities and agricultural sources. In future studies, the relationship between efficacy of plasmid transfer and the genetic features of the plasmids will be scrutinised, for this information will be relevant not only for epidemiologists and Ethiopian surveillance but also for the global scientific community to associate the success of the spread and dissemination of an antibiotic resistance gene with a plasmid type and mobility.
In conclusion, we report a high prevalence of IncF-like plasmids that might be involved in the mobilization of CTX-M and other AMR genes in Ethiopia. Mostly identified from the international successful ST131 lineage, these plasmids harbour the ISEcp1 element for effective gene capture as well as multiple addiction systems to select for plasmid maintenance in daughter cells. The data indicate the underlying molecular basis for the previously reported extensive prevalence of bla CTX-M-15 in Ethiopia 11 . Knowledge of the role of transmissible plasmids in the spread of AMR genes among extra-intestinal E. coli populations in Ethiopia is an important step. Not only will it help strengthen national infection prevention and control systems, but it also opens up the possibility of developing therapeutic strategies to target bacteria harbouring such plasmids to limit their subsequent acquisition and transmission of AMR within bacterial populations 54,55 . Overall, the data represent high AMR plasmid carriage among CTX-M ESBL-producing E. coli isolates from four facilities in Ethiopia. As a result, the potential for plasmid transmissibility is very high, as is the likelihood of further rapid spread of AMR genes of diverse families.

Methods
Study design and samples. The isolates were retrieved from a biobank after having been collected in 2018 from four geographically distinct facilities as part of ongoing national AMR surveillance initiative. The initiative was launched in 2017 by the EPHI under the supervision of the Ministry of Health and operates under the auspices of the World Health Organization Global AMR and Use Surveillance (GLASS) initiative (https:// www. who. int/ initi atives/ glass). Detailed sample collection procedure including patient inclusion and exclusion criteria is reported elsewhere 11,56 and strictly adhered to standard practices for clinical microbiological sampling established by the Ohio State University Global One Health initiative 57 . All in the biobank are clinical isolates and not isolates from the general population or other epidemiologic scenarios.
Initial phenotypic characterization, strain screening, phylo-typing as well as β-lactamase gene detection and antibiotic susceptibility testing were reported for 204 ESBLs-producing E. coli clinical isolates in our recent study 11 . In the current investigation, we considered 100 CTX-Ms-producing isolates obtained from urine (n = 84), pus (n = 10) and blood (n = 6) from the original set. It was sufficient to focus on just 100 isolates because all the CTX-Ms-encoding gene types identified in our initial study were included within this sub-collection. However, the over-representation of isolates from urine suggests that most of these strains will be enriched for virulence factors associated with genitourinary invasion. Moreover, we have no data on the antimicrobial exposure at the time of collection. This can be relevant because it is conceivable that certain patients from whom the isolates were obtained were receiving antimicrobial therapy, creating potential for bias toward over-representation of certain antimicrobial resistance genes.
Ethics approval and consent to participate. The study was approved by the EPHI Scientific and Ethical Review Board (EPHI-IRB-054-2017) and the College of Natural and Computational Sciences Institutional Review Board, Addis Ababa University (CNS-IRB/039/2019). As all bacterial isolates included in this study were sourced from a biobank, the study did not directly involve patients, human material, or personal data identifiers.
Plasmid transmissibility testing. Plasmids were transferred by either the conjugation or chemical transformation method. Conjugation was performed by a mating assay using E coli J53 AziR as recipient strain 58 . Trans-conjugants were selected on Luria-Bertani agar (LA) plates containing sodium azide (150 µg/mL) and cefotaxime (2 µg/mL). This necessitated that all donor strains were pre-tested for susceptibility to sodium azide and E coli J53 AziR was pre-tested for susceptibility to cefotaxime. If plasmids were non-conjugative, chemical transformation was performed using the recipient strain E. coli HB101 (Promega, Sweden). Plasmids were purified using the GeneJET plasmid miniprep kit (Thermo Fisher Scientific Inc.) and transformed into the chemically competent recipient strain using heat shock at 42 °C. Transformants were selected on LA plates supplemented with cefotaxime (2 µg/mL).

ESBLs-gene detection and antibiotic susceptibility.
Isolates were analysed for the presence of ESBL-encoding genes bla TEM, bla SHV, bla CTX-M and bla OXA using a combination of established PCR and sequencing methods as previously described 59 . Purified PCR products were sequenced using the service of Eurofins genomics (Ebersberg, Germany). The β-lactamase gene types were identified by alignment with sequences in GenBank using BLAST (http:// www. ncbi. nlm. nih. gov/ BLAST). Antibiotic susceptibility testing was done for PCR-based detection of ISEcp1 element. Detection of the insertion sequence ISEcp1 was determined by PCR using the combination of ISEcp1 primer and CTX-M reverse consensus primer (MA1 reverse) as previously described 61 . An amplified product is indicative of the ISEcp1 element situated upstream of the bla CTX-M genes. The PCR products were purified and confirmed by sequencing.

Plasmid transmissibility and biofilm formation. Biofilm formation was determined for 12 parental
isolates. The isolates chosen and their detected genotypes and phenotypes are listed in Supplementary Table S1. All parental isolates belonged to the known extra-intestinal pathogenic E. coli (phylogenetic group B2 and D). All except isolate 149 are the international high-risk clone ST131. All produce at least one CTX-M type, and all except isolate 74 exhibited the detection of more than one IncF plasmid replicon type. The measurement of biofilm forming capacity of the parental isolates and their corresponding recipients used a previously described protocol with slight modification 62 . Briefly, both groups of bacteria were grown in M63 minimal media with glycerol as the carbon source, and with respective antibiotics overnight at 37 °C with aeration. Cefotaxime (2 µg/ml) was used for donor parental strains and 2 µg/ml cefotaxime and 100 µg/ml sodium azide were used for recipients.
The strain E. coli J53 AziR was used as a control and grown in the M63 medium containing 100 µg/ml sodium azide. From overnight bacterial cultures, 3 µl aliquots were mixed with in 147 µl fresh M63 medium in the wells of a sterile 96-well round-bottom µl dish and incubated overnight at 37 °C. Developed biofilms were heat-fixed and stained with 0.1% w/v crystal violet solution. The stained biomass was recovered by solubilisation in 33% (v/v) glacial acetic acid. The extent of solubilized biofilm was then recorded spectroscopically at an absorbance of 560 nm, and the efficiency of biofilm formation calculated by normalization with planktonic growth recorded as the optical density at a wavelength of 600 nm. The specific biofilm formation (SBF) was determined using the formula SBF = (AB-CW)/G, where AB is OD 560 of stained cells, CW is OD 560 of control wells cultured with M63 medium only, and G is the OD 600 of the bacteria growth calculated from G = OD 600 (24 h)-OD 600 (0 h). The strains were categorized as weak biofilm former (SBF ≤ 0.5), moderate biofilm former (SBF = 0.5-1.0) and strong biofilm former (SBF ≥ 1.0). For logistical reasons, the sample size was restricted to 12 to enable a minimum of three biological replicates with three technical repeats to ensure data quality. Furthermore, the selection process ensured that the parental isolates represented the different phylogenetic backgrounds.
Serum resistance measurements. Serum sensitivity was determined for 10 parental isolates and their corresponding trans-conjugants using a previously described protocol 49 . Briefly, the strains were grown in LB broth overnight at 37 • C with aeration. Five µl of the overnight cultures were sub-cultured into 495 µl fresh LB broth and grown statically for 2 h at 37 • C. Following centrifugation at 7600 g for 3 min, pellets were resuspended in 500 µl phosphate-buffered saline. Volumes of 20 µl from the washed bacteria were mixed with 180 µl of normal human serum in a 96-well flat bottom microtiter dish and incubated statically at 37 °C for 3 h. At 0 h and 3 h time points, 20 µl was removed from the wells and plated after suitable serial dilution on LB plates containing 2 µg/ml cefotaxime for parental strains, 2 µg/ml cefotaxime and 100 µg/ml sodium azide for trans-conjugants, and 100 µg/ml sodium azide for the E. coli J53 AziR control. The number of colony forming units (CFUs) of bacteria was determined after the plates were incubated overnight at 37 °C. Susceptibility to active serum was calculated as follows: log kill = (log10 CFU/µl of initially added bacteria-0 h)-(log10 CFU/µl of bacteria surviving the incubation after 3 h) according to a previous report 63 . All experiments were conducted in duplicate. The selection process of the 10 isolates ensured that the parental isolates represented the different phylogenetic backgrounds. The recipient J53 strain alone was used as a control in all assays. www.nature.com/scientificreports/ Statistical analysis. The data was prepared using Excel spread sheets (Microsoft Office) and imported to SPSS version 20.0. The frequencies of different variables were calculated. Cross-tabulation and graphs were used to present the different relation between data.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.